Proteins, also referred to as polypeptides, are known to exhibit various pharmacologic actions in vivo. Thanks to advances in genetic engineering and cell engineering technologies, some have been produced in large mounts for pharmaceutical application using organisms such as Escherichia coli, yeasts, animal cells and hamsters. Such protein pharmaceuticals include interferons (alpha, beta, gamma), interleukin 2, erythropoietin and granulocyte colony-stimulating factor (G-CSF). These proteins, however, since they have generally a short biological half-life, must be administered frequently, posing the significant physical burden of injection on patients. To solve this problem, various attempts have been made to develop sustained-release preparations. Since proteins represented by cytokines must be administered with great care while monitoring their therapeutic effect, there is a need for development of injectable sustained-release preparations, particularly microcapsular sustained-release preparations, that have a release duration of about 1 to 2 weeks. It is generally known, however, that proteins undergo denaturation and lose their bioactivity upon exposure to heat, organic solvents, strong shearing force etc. For example, an aqueous solution of a protein can rapidly lose its bioactivity when heated at 60.degree. C. for 20 minutes. Bioactivity of a protein can decrease upon heating, even at a lower temperature of 50.degree. C. for about 1 hour. Similarly, protein bioactivity is known to decrease in the presence of an organic solvent such as ethanol or dichloromethane.
WO93/06872 discloses a technology for preparing a pharmaceutical preparation comprising porous particles of a biodegradable polymer allowing osteogenic proteins to be released over an extended period of time; an osteogenic protein and autologous blood aggregate. In this technology, the active ingredient osteogenic protein is adsorbed to the particles just before administration, and autologous blood is added to form an aggregate, to control release. Sustained-release duration is about several weeks. This method is not commonly usable because it involves the use of autologous blood.
In the Journal of Controlled Release, Vol. 23, p. 157 (1993), A. Supersaxo et al. describe a technology in which a porous microsphere is prepared using a biodegradable polymer, after which it is permeated with a macromolecule, is permitted to permeate therein to incorporate it in the microsphere without bringing the macromolecule into contact with an organic solvent. Specifically, since the polylactic acid used is hydrophobic, 50% ethanol (an organic solvent) is first used to wet the microsphere. The ethanol is then replaced with water and then with a solution of a macromolecule.
Japanese Patent Unexamined Publication No. 32559/1993 (EP-A 473268) discloses a method of producing a pharmaceutical composition by dissolving pharmaceutical composition components and a bioactive substance in an organic solvent or uniformly dispersing pharmaceutical composition components and a bioactive substance in an organic solvent or aqueous medium, and then drying the solution or dispersion.
Although various attempts have been made to produce sustained-release preparations retaining the bioactivity of proteins etc., as stated above, no satisfactory sustained-release preparations have been obtained as to efficiency of drug permeation into matrix, suppression of initial drug burst, constant long-term drug release etc.